The present invention relates to the field of nuclear receptors. Specifically, the present invention is based in part on the isolation of DNA encoding RX receptors and on the novel observation that two different types of nuclear receptors, retinoic acid receptors (RAR) and thyroid receptors (TR) dimerize with RX receptor (RXR) to form a heterodimer. The heterodimer is capable of binding to retinoic acid response elements (RARE), thyroid receptor response elements (TRE), or RX response elements (RXRE) at physiological conditions. Based on this observation, the present invention provides methods of identifying agents capable of binding the disclosed heterodimers, as well as identifying DNA sequences capable of being bound by the heterodimers. In addition, the present invention describes a method to identify mammalian-specific enzymes involved in RA metabolism, novel heteromeric partners of RXR and co-factors involved in the activation function of retinoic acid receptors.
Retinoids are metabolites of vitamin A (retinol) which are thought to be important signaling molecules during vertebrate development and for controlling the differentiation state of several adult tissues (for reviews see Brockes, Neuron 2:1285-1294 (1989) and Brockes, Nature 345:766-768 (1990); Sherman, Retinoids and Cell Differentiation, Sherman, M. I. (ed). CRC Press (1986); Summerbell et al., Trends in Neurosci. 13:142-147 (1990)). Two families of nuclear retinoid receptors have been characterized. Retinoic acid receptors, which include RAR-xcex1, RAR-xcex2 and RAR-xcex3 (for reviews see Ruberte et al., Development 111:45-60 (1991b) and Chambon et al., Seminars in Dev. Biol. 2:153-159 (1991a)), have a high affinity for all-trans retinoic acid (RA) and belong to the same class of nuclear receptors as thyroid hormone (TRs), vitamin D3 (VDR) and ecdysone (EcR) receptors (see Koelle et al., Cell 67:59-77 (1991)). Members of the RXR family, RXR-xcex1 (Mangelsdorf et al., Nature 345:224-229 (1990) herein incorporated by reference), RXR-xcex2 (Hamada et al., Proc. Natl. Acad. Sci. USA 86:8289-8293 (1989) herein incorporated by reference) and RXR-xcex3 respond to much higher concentrations of RA, and the natural ligand for RXRs appears to be a new stereoisomer of RA. RXRs belong to a different class of nuclear receptors which includes the Drosophila ultraspiracle (usp) gene product (Oro et al., Nature 347:298-301 (1990)).
Synthetic and natural DNA response elements (REs) have been characterized for TRs (Glass et al., Nature 329:738-741 (1987); Umesono et al., Cell 65:1255-1266 (1991) and see refs therein), RARs (Vasios et al., Proc. Natl. Acad. Sci. USA 86:9099-9103 (1989) and Vasios et al., EMBO J. 10:1149-1158 (1991); de Thxc3xa9 et al., Nature 343:177-180 (1990); Leroy et al., Proc. Natl. Acad. Sci. USA 88:10138-10142 (1991a) and refs therein), and RXRs (Mangelsdorf et al., Cell 66:555-561 (1991)). All of these REs consist of the repetition of a core motif, PuGTGTCA (Pu=purine) (or a related sequence), in different configurations with respect to both the orientation (direct or inverse repetition) and the spacing of the two motifs. The recognition of REs by a given receptor appears to be dependent on the actual sequence, orientation and spacing of the repeated motifs. Systematic studies of the influence of the spacing between directly repeated motifs have shown that RARs have a preference for 5 bp spaced motifs (Umesono et al., Cell 65:1255-1266 (1991)), whereas TRs and RXRs preferentially recognize motifs separated by 4 bp (Umesono et al., Cell 65:1255-1266 (1991)) and 1 bp (Mangelsdorf et al., Cell 66:555-561 (1991)), respectively. The presence of repeated motifs in these REs, and the demonstration that the glucocorticoid and oestrogen receptors bind as dimers to palindromic REs made up of similar motifs (Schwabe et al., Trends Biochem. Sci. 116:291-296 (1991); Luisi et al., Nature 352:497-505 (1991); and references therein) have suggested that RARs, TRs and RXRs also bind as dimers to REs. This possibility has been directly supported by in vitro binding evidence in the case of TRs and RARs (Glass et al., Cell 59:697-708 (1989); Glass et al., Cell 63:729-738 (1990); Lazar et al., Mol. Cell. Biol. 11:5005-5015 (1991); Forman et al., Gene 105:9-15 (1991)). However, it has also been reported that the in vitro binding of RAR (Glass et al., Cell 63:729-738 (1990)) and TR (Murray et al., Mol. Endocrinol. 3:1434-1442 (1989); Burnside et al., J. Biol. Chem. 265:2500-2504 (1990)) to REs can be greatly stimulated by the addition of, as yet, uncharacterized factor(s) present in nuclear extracts of a variety of cells. Furthermore, evidence has been presented indicating that these factors may form heterodimers with RAR (Glass et al., Cell 63:729-738 (1990)) and TR (Lazar et al., Mol. Cell. Biol. 11:5005-5015 (1991); Nxc3xa4xc3xa4r et al., Cell 65:1267-1279 (1991)), which bind with greater affinity to REs than the isolated receptors.
In the course of purification of RARs overexpressed in a variety of host-vector systems, these receptors lose the capability to bind the RA response element (RARE) of the RAR-xcex22 promoter (xcex2-RARE) (de Thxc3xa9 et al., Nature 343:177-180 (1990); Sucov et al., Proc. Natl. Acad. Sci. USA 87:5392-5398 (1990); Mendelsohn et al., Development 113:723-734 (1991)), which could be recovered by addition of HeLa cell nuclear extracts, irrespective of the source of over expressed RARs.
Retinoids have been used in the treatment of actinally aged skin (Ellis et al., Pharmacol. Skin. 3:249-253 (1989)), various types of dermatoses (Gollnick, Dermatological 175(1): 182-195 (1987)), disorders of keratinization (Happle et al., Dermatological 175(1):107-124 (1987)), rheumatoid arthritis (Brinckerhoff et al., 1985 Retinoids, Differentiation and Disease, Pitman, London (Ciba Foundation Symposium 113) p. 191-211), basal cell carcinoma (Peck, Dernatological 175(1):138-144 (1987)), and systemic sclerosis (Maurice et al., Pharmacol. Skin. 3:235-239 (1989)). In addition, retinoids have been demonstrated to possess immunostimulating activity (Dennert, 1985 Retinoids, Differentiation and Disease, Pitman, London (Ciba Foundation Symposium 113) p. 117-131), inhibit epidermal terminal differentiation (Lichti et al., 1985 Retinoids, Differentiation and Disease, Pitman, London (Ciba Foundation Symposium 113) p. 77-89), modulate carcinogenesis in the urinary bladder (Hicks et al., 1985 Retinoids, Differentiation and Disease, Pitman, London (Ciba Foundation Symposium 113) p. 168-190), regulate differentiation in embryonal carcinoma cells (Sherman et al., 1985 Retinoids, Differentiation and Disease, Pitman, London (Ciba Foundation Symposium 113) p. 42-60), regulate differentiation in tracheal epithelial cells (Jetten et al., 1985 Retinoids, Differentiation and Disease, Pitman, London (Ciba Foundation Symposium 113) p. 61-76), inhibit neoplastic transformation (Bertram et al., 1985 Retinoids, Differentiation and Disease, Pitman, London (Ciba Foundation Symposium 113) p. 29-41), possess anti-inflammatory activity (Ney et al., Dermatological 175(1):93-99 (1987)), modulate melanoma growth (Amos et al., Pharmacol. Skin. 3:29-36 (1989)), and may play an important role in cholesterol metabolism (Rottman et al., Mol. Cell. Biol. 11:3814-3820 (1991)).
It is unknown what the molecular basis is for the various effects retinoids are able to regulate. One possibility is that the various effects regulated by retinoids are caused by the interactions of the retinoid ligand with a tissue specific RAR receptor. Alternatively, the various receptors may bind to different RE motifs with differing affinities.
Using the observations disclosed in the present invention, it is now possible to examine the interactions of retinoids, or derivatives thereof, with specific RAR/RXR, and TR/RXR heterodimeric combinations. Additionally, each of the heterodimeric combinations can be examined for it""s affinity for different RARE, TRE or RXRE sequences. Such a system will lead to a better understanding of the biological effects stimulated by retinoids and lead to the identification of the next generation of retinoids.
The present invention is based in part on the novel observation that three types of nuclear receptors, RAR, RXR and TR, can form heterodimers at physiological conditions which possess a greater affinity for binding to the various RE motifs than each of the respective homodimers.
Based on this observation the present invention provides heterodimeric proteins which are comprised of two subunits, one of the subunits is either a RAR or TR, and the other subunit is a RXR.
The present invention further provides highly purified subtypes of RXR. The highly purified forms of RXR have a specific activity from about 1461 to 7,750,000 cpm/xcexcg. Examples of the amino acid sequences of various RXR""s of the present invention are depicted in Sequence ID No. 2 (mRXR-xcex2), Sequence ID No. 4 (hRXR-xcex2), Sequence ID No. 6 (mRXR-xcex1), and Sequence ID No. 8 (mRXR-xcex3). The RXR""s of the present invention includes monomers and multimers (such as homodimers) of each of the isoforms and subtypes of RXR.
The present invention further provides methods of purifying subtypes and isoforms of RXR. In detail, RXR""s can be purified by:
a) contacting a sample containing a RXR protein with a DEAE chromatography column in the presence of a buffer containing from about 50 mM KCl;
b) retrieving the RXR in the flow through fraction from the column;
c) contacting the flow through fraction (b) with a HEP-UG column;
d) eluting the RXR from the column using from about 290 mM KCl;
e) contacting the KCl eluted RXR (d) with a phenyl-5PW column;
f) eluting the RXR from the column using from about 250 mM ammonium sulfate;
g) contacting the ammonium sulfate eluted RXR (f) with a HEP-TSK column;
h) eluting the RXR from the column using from about 250 mM KCl;
i) contacting the KCl eluted RXR (h) with a HAP-TSK column; and
j) eluting the RXR from the column using from about 150 mM potassium phosphate.
The present invention further provides DNA sequences which are capable of binding to any one of the above heterodimers or homodimers.
The present invention further provides antibodies which are capable of binding to any one of the above heterodimers or homodimers.
The present invention further provides methods of identifying agents capable of binding to the heterodimers of the present invention comprising the steps of:
1) incubating an agent with one of the heterodimers of the present invention; and
2) determining whether the agent bound to the heterodimer.
The present invention further provides methods of identifying agents capable of inducing transcription of a sequence operably linked to an RARE, TRE, or RXRE. These methods comprise the steps of:
1) incubating a cell, organism, or extract thereof, which has been altered to express one or more of the dimers described herein, with an agent, wherein said cell or organism contains a reporter sequence operably linked to an RARE; and
2) assaying for the expression of the reporter sequence.
The present invention also provides methods of directing the expression of a DNA sequence in response to a specific agent by first identifying a dimer/RE/agent combination which is capable of inducing transcription in the above described assay and then altering a cell or organism such that it will express the heterodimer and contains a DNA sequence, operably linked to the RE.
The present invention further provides DNA sequences which encode members of RXR family of receptors. Specifically, sequences encoding 1 isoform of mRXR-xcex2 (Sequence ID No. 1), 1 isoform of hRXR-xcex2 (Sequence ID No. 3), 1 isoform of mRXR-xcex1 (Sequence ID No. 5), and 1 isoform of mRXR-xcex3 (Sequence ID No. 7), are described.
FIGS. 1A/B, C, D, and E. Dependence of hRAR-xcex3 DNA binding on HeLa and insect cell RBF(s); Purification of a HeLa cell RBF
FIGS. 1A and 1B. Gel retardation assays in which increasing amounts of HeLa NE (0, 1.5, 3 and 6 mg of protein) were added to incubations containing the xcex2-RARE probe and crude (lanes 1-4) or purified (lanes 5-8) rVV-expressed hRAR-xcex3, crude (lanes 9-12) or purified (lanes 13-16) rBV-expressed hRAR-xcex3 in-vitro translated receptor (lanes 20-23) and bacterially-expressed hRAR-xcex3 (lanes 24-27). Arrows indicate positions of HeLa- and Sf9-cell specific complexes. The binding of two independent preparations of HeLa nuclear extract in the absence of hRAR-xcex3 is shown in lanes 17-19 and 28-30.
FIG. 1C. Gel retardation assays in which bacterially-expressed hRAR-xcex3 (10 fmols) was incubated with extracts prepared from HeLa (3 xcexcg of protein), Sf9 (5 xcexcg) and Drosophilia S2 (5 xcexcg) cells as indicated. Lanes 5-7 represent binding of these extracts in the absence of hRAR-xcex3.
FIG. 1D. Silver stained gel representing each step of HeLa cell RBF purification. The amounts of protein (in xcexcg) loaded in each lane is: WCE, 1.5; DEAE FT, 1.1; HEP-UG, 2; Phenyl-5PW, 5; HEP-TSK, 1.3 and HAP-TSK, 0.05 (estimate). The migration of molecular weight standards (BioRad) is indicated.
FIG. 1E. Gel retardation assay representing each step of HeLa cell RBF purification. HeLa NE (4 xcexcg of protein) was included as a positive control (lane 2). The amount of HeLa cell protein (in xcexcg) used in each lane is: WCE, 10; DEAE FT, 0.5; HEP-UG, 0.18; Phenyl-5PW, 0.12; HEP-TSK, 0.03 and HAP-TSK 0.001 (estimate). Lanes contain the stated amounts of protein from each purification step and xcx9c10 fmols of bacterially-expressed hRAR-xcex3 where indicated.
FIGS. 2A-2B. Amino acid alignment of hRXR-xcex2 with mRXR-xcex1, -xcex2 and -xcex3.
Amino acid identity is indicated by a star (between all RXRs) and/or a dot (between human and mouse RXR-xcex2). Based on homology with other nuclear receptors, the DNA (region C) and ligand binding (region E) domains are indicated. The amino acid sequences of seven peptides obtained by tryptic digestion of purified HeLa cell RBF are indicated in shaded boxes. Open headed arrows on p24 and p27 indicate amino acids from which PCR primers were deduced (see text). Note that p25 and p28 were the only peptides obtained which could discriminate between members of the RXR family. Genbank accession numbers for the nucleotide M84817, M84818, M84819, M84820 sequence of mRXR-xcex1, -xcex2 and -xcex3 and hRXR-xcex2 are respectively:
FIGS. 3A and B. Cloned RXRs stimulate binding of hRARs to the xcex2-RARE
FIG. 3A. Approximately 10 fmols of bacterially-expressed hRAR-xcex3 were incubated with the xcex2-RARE in the absence (lane 1) or presence of equal molar amounts of in vitro translated mRXRs as indicated. As a control, a volume of rabbit reticulocyte lysate (RRL) corresponding to that which contained mRXRs (2 xcexcl) was also mixed with hRAR-xcex3 (lane 5). Binding of RXRs and RRL to the xcex2-RARE in the absence of hRAR-xcex3 is shown in lanes 6-9.
FIG. 3B. RARs (xcx9c10 fmols of each receptor) were prepared by in vitro translation and incubated with the xcex2-RARE in the absence or presence of either HeLa nuclear extract (3 xcexcg of protein) or mRXR-xcex1 (xcx9c10 fmols, translated in vitro). Control lanes 10-12 correspond to incubation of HeLa NE, mRXR-xcex1 and RRL, respectively, with the xcex2-RARE in the absence of RARs.
FIGS. 4A and B. Binding of hRAR-xcex3, mRXR-xcex1 and chicken TRxcex11 to the xcex2-RARE and TREpal.
FIG. 4A. Binding of isolated RAR and RXR to xcex2-RARE and TREpal probes. Lanes 1-9 and 10-18 (xcex2-RARE binding) are derived from the same gel; however, the latter lanes have been exposed for a longer period of time to visualize weak complexes. Similarly, lanes 19-26 and 27-34 (TREpal binding) are identical except that the latter lanes were exposed longer for the same purpose. Gel retardation assays in which partially purified (DEAE FT, 2 xcexcg), or purified (HAP-TSK,1 ng) HeLa cell RBF, mRXR-xcex1 (xcx9c10 fmols translated in vitro), mRXRxcex1ER(F) (xcx9c10 fmols translated in vitro) and bacterially-expressed hRAR-xcex3 (xcx9c10 fmols) were incubated with the xcex2-RARE (the sequence of the upper strand of this probe is given) as indicated. In the presence of specific antibodies, mRXRxcex1ER(F) (lanes 6 and 15) and hRAR-xcex3 (lanes 9 and 18) supershifted complexes were observed, whereas no mRXRxcex1ER(F) complex was supershifted with a non-specific antibody (Ab4xcex3, lanes 5 and 14). Lanes 19-34 correspond exactly to lanes 1-18 with regard to sample contents but the former lanes represent binding to a TREpal (the sequence of the upper strand of this probe is given). Arrows denote specific complexes.
FIG. 4B. Cooperative binding of RXR and either RAR or c-erbA to xcex2-RARE and TREpal probes. Note that the exposure time of these gels was identical to that of lanes 1-9 and 19-26 in part A. Lanes 1-9 represent bacterially-expressed hRAR-xcex3 (xcx9c10 fmols) binding to the xcex2-RARE alone (lane 1) or in the presence of DEAE-FT or HAP-TSK preparations of HeLa cell R0F, c-erbA (xcx9c10 fmols translated in vitro), mRXR-xcex1, mRXRxcex1ER(F) or RRL as indicated. The hRAR-xcex3/mRXRxcex1ER(F)/xcex2-RARE complex (lane 6) was supershifted by both Ab4-xcex3 (lane 7) and AbF3 (lane 8). Lanes 10-17 depict c-erbA (xcx9c10 fmols, translated in vitro) binding to the xcex2-RARE alone (lane 10) or in the presence of DEAE FT or HAP-TSK preparations of HeLa cell RBF, hRAR-xcex3, mRXR-xcex1, mRXRxcex1ER(F) or RRL as indicated. A c-erbA/mRXRxcex1ER(F)/xcex2-RARE supershifted complex was observed with addition of AbF3 (lane 16). Lanes 18-34 correspond exactly to lanes 1-17 except the former lanes represent cooperative binding interactions on the TREpal. Arrows denote specific complex formation. Amounts of receptors were as indicated in part A.
FIG. 5. Chemical crosslinking and co-immunoprecipitation of RAR and RXR
FIG. 5A. [35S]hRAR-xcex3 (xcx9c50 fmols) was incubated with an equal molar amount of either unlabeled hRAR-xcex3 or mRXRxcex1ER(F) as indicated. Incubations were carried out in the absence or presence of 500 fmols of xcex2-RAREwt and DSS (final concentration of 1 mM) as indicated. The upper arrow denotes the cross-linked product observed in lanes 3, 5, 7 and 9 and the lower arrow corresponds to the [35S]hRAR-xcex3 monomer. Lane 1 is a reference lane for the migration of [35S]hRAR-xcex3.
FIG. 5B. Part B, is similar to A, except that the labeled receptor is [35S]mRXRxcex1ER(F). Crosslinked complexes and [35S]mRXRxcex1ER(F) monomers are indicated by the upper and lower arrows, respectively. Additions of unlabeled receptors, xcex2-RARE, DSS and precipitating antibody are as indicated. Lane 1 is a reference lane for migration of [35S]mRXRxcex1ER(F).
FIGS. 6A and B. Characterization of RAR/RXR interaction by deletion and mutational analysis of each protein
FIG. 6A. DNA binding of hRAR-xcex3 mutants to the xcex2-RARE. hRAR-xcex3 mutants were translated in vitro (using RRL) and the amount of translated protein normalized (xcx9c10 fmol/assay) as described (see experimental procedures). Gel retardation assays were carried out in the presence or absence of an equal molar amount of mRXR-xcex1 as indicated. After autoradiography, specific complexes were excised and counted in a scintillation counter to quantify the extent of complex formation by each mutant. Shown is a schematic diagram of the mutants and the corresponding activity relative to hRAR-xcex3 wt.
FIG. 6B. Binding of mRXR-xcex1 mutants to the xcex2-RARE was similarly assessed. mRXR-xcex1 mutants were translated in vitro and the gel retardation assay carried out in the presence or absence of an equal molar amount (xcx9c10 fmols) of bacterially-expressed hRAR-xcex3. The experiment was quantified as described in part A. Note that the DNA binding properties of one mutant, mRXR-xcex1xcex94449-466, are not shown in the gel but the complex formed in the presence of this mutant and hRAR-xcex3 was indistinguishable from that of mRXR-xcex1 or mRXRxcex1xcex94455-466.
FIG. 7. Comparison of RAR/RXR complex formation on direct repeats of different inter-repeat spacing
xcx9c10 fmols of bacterially-expressed hRAR-xcex3, in vitro translated hRAR-xcex3-xcex94C4, mRXR-xcex1wt or mRXR-xcex1xcex94C4 were incubated in various combinations as indicated with the xcex2-RARE (lanes 1-8) or xcex2-RARE1 (lanes 9-16) probes (the repeated motifs of each are given). The right panel shows a lesser exposure of lanes 1-3. Specific complexes are indicated by the arrow. Other bands present are due to non-specific E. coli or RRL proteins (data not shown).
FIG. 8. Binding of Cos cell-expressed mRXR-xcex1 to the xcex2-RARE
Gel retardation assays in which an aliquot (10 xcexcg of protein) of Cos cell WCE, prepared from cells transfected with mRXR-xcex1 (lanes 5-12) or the parental expression vector (pSG5, lanes 1-4) was incubated with the xcex2-RARE in the presence or absence of anti-RAR monoclonal antibodies Ab9xcex1, Ab7xcex2 and Ab4xcex3 as indicated. The upper and lower arrows denote positions of supershifted and non-supershifted complexes, respectively.
FIG. 9. Amino acid sequence alignment of a subdomain implicated in the dimerization potential of selected nuclear receptors
Region of the mouse estrogen receptor (MER) which is critical for (homo)dimerization (Fawell et al., Cell 60:953-962, stars indicate residues which when mutated destroyed the homodimerization potential of the receptor). Residues contained in shaded boxes correspond to heptad repeat 9 proposed by Forman et al., Mol. Endocrinol. 4:1293-1301 (1990). In the case of mRXR-xcex1, usp and E75A, another heptad (underlined) repeat is possible. Proline residues, which may impart flexibility to this region are also indicated. The sequences shown are from the following references: mER, (White et al., Mol. Endocrinol. 1:735-744 (1987) (see also Fawell et al., Cell 60:953-962 (1990)); hRAR-xcex3, Krust et al., Proc. Natl. Acad. Sci. USA 86:5310-5314 (1989); cTR-xcex11, Sap et al., Nature 34:635-640 (1986); hVDR, Baker et al., Proc. Natl. Acad. Sci. USA 85:3294-3298 (1988); EcR, Koelle et al., Cell 67:59-77 (1991); mRXR-xcex1, the current report; usp, Oro et al., Nature 347:298-301 (1990); E75A, Segraves et al., Genes and Dev. 4:204-219 (1990); svp, Mlodzik et al., Cell 60:211-224 (1990); rNGFI-B, Milbrandt, Neuron 1:183-188 (1988); hear-1, Miyajima et al., Cell 57:31-39 (1989).
FIGS. 10A, B, and C. Chimeric RARxcex1 and RXRxcex1 activate transcription autonomously in yeast
FIG. 10A, Schematic representation of chimeric RARxcex11-ER.CAS and RXRxcex1-ER(C) receptors and their truncated derivatives. Numbers refer to amino acid positions. The chimeric receptors contain the DBD of human ER (hatched box) and were expressed from the constitutive yeast PGK (phosphoglycerate kinase) gene promoter in the 2xcexc-derived yeast multicopy plasmids YEp10 and YEp90 (Pierrat, B. et al., Gene 119:237-245 (1992)). FIGS. 10B1 and 10B2, Dose responses of RARxcex11-ER. CAS and RXRxcex1-ER(C) to all-trans retinoic acid (T-RA) and 9-cis retinoic acid (9C-RA). The chimeric receptors RARxcex11-ER.CAS and RXRxcex1-ER(C) were expressed in a reporter strain (PL3) which contains a chromosomally integrated 3ERE-URA3 reporter gene (Pierrat, B. et al., Gene 119:237-245 (1992)), in the presence of T-RA or 9C-RA at the concentrations indicated. Transcription of the reporter gene was determined by measuring the specific activity of the URA3 gene product OMPdecase (orotidine-5xe2x80x2-monophosphate decarboxylase), and is represented as fold induction above the level of OMPdecase activity observed in the absence of ligand. FIGS. 10C1 and 10C2, Retinoic acid derivatives differentially induce transactivation by RARxcex11-ER.CAS and RXRxcex1ER(C). Induction of reporter activity in the yeast strain PL3 expressing RARxcex11-ER.CAS or RXRxcex1-ER(C) in the presence of the following ligands; T-RA, 9C-RA, T-ddRA (all-trans 3,4-didehydroretinoic acid), 9C-ddRA (9-cis 3,4-didehydroretinoic acid). The final concentration of ligand was 10xe2x88x926 M. The level of transactivation of the reporter gene is given as units of OMPdecase activity (nmoles of substrate transformed/min/mg protein).
FIGS. 11A and B. RARxcex1 and RXRxcex1 cooperate to activate a RARE reporter gene in yeast.
FIG. 11A, Schematic representation of the promoter region of the DR5-URA3 reporter used in transactivation experiments. URA3 promoter sequences required for both basal and activated transcription were deleted and replaced with a RARE sequence. This element consists of a direct repeat of the motif 5xe2x80x2-AGGTCA-3xe2x80x2 separated by five base pairs (DR5; the sequence of which is shown). The position of the DR5 and the TATA box relative to the ATG start codon (+1) are also shown, and the bent arrow indicates the approximate site of initiation of transcription of the URA3 mRNA. The reporter gene was maintained in yeast strain YPH250 on a centromeric plasmid. FIG. 11B, RXRxcex1 enhances RARE activity on a DR5 element in yeast. OMPdecase activities measured in extracts of transformants containing the DR5-URA3 reporter plasmid and multicopy plasmids (see FIG. 10) expressing RARxcex1, RARxcex1dnxcex94AB, RXRxcex1, RXRxcex1dnxcex94AB in the combinations indicated, or parental vectors (control), in the presence or absence of ligand (T-RA or 9C-RA; final concentration 5xc3x9710xe2x88x927 M). In RARxcex1dnxcex94AB and RXRxcex1dnxcex94AB, the A/B regions and part of the C-terminus of RARxcex1 and RXRxcex1, respectively, have been deleted, resulting in transcriptionally inactive mutant derivatives (data not shown). The mean values and standard deviations presented are derived from at least two experiments using three separate transformants for each clone. FIGS. 11C1 and 11C2, 9C-RA induces RXRxcex1 activity on a DR5-reporter gene in yeast. Dose responses to T-RA and 9C-RA in transformants containing the DR5-URA3 reporter plasmid, and coexpressing RARxcex1 with RXRxcex1 or RXRxcex1dn as shown. RXRxcex1dn is a dominant negative receptor in mammalian cells and contains a C-terminal deletion (Durand, B. et al., Cell 71:73-85 (1992)). Transactivation is represented as fold induction of reporter activity above the value obtained in the absence of ligand.
FIG. 12. RARxcex1 and RXRxcex1 produced in yeast cooperate for DNA-binding to a RARE in vitro.
Gel retardation assays were performed using a labelled DR5 probe (the sequence of which is indicated below the figure) and cell-free extracts prepared from yeast transformants expressing no receptor (lane 1), mRXRxcex1 (mouse RXRxcex1 receptor) (lane 3), hRARxcex1 (human RARxcex11 receptor) (lane 5), hRARxcex1 and mRXRxcex1 (lane 7), hRARxcex1 and mRXRxcex1dn (mouse RXRxcex1dn receptor) (lane 9). Specificity of binding was verified by supershifting retarded complexes with the RARxcex1-specific monoclonal antibody Ab9a (Gaub, M. P. et al., Exp. Cell Res. 201:8:335-346 (1992)). Lanes 2, 4, 6, 8 and 10 contain identical samples as lanes 1, 3, 5, 7 and 9 respectively, but the samples were incubated with antibody immediately before electrophoresis. Arrows indicate the specific and supershifted complexes.
FIGS. 13A, B, and C. Direct Repeats of a Half Site Motif with Different Spacings and an Inverted Repeat of the Same Sequence with No Spacing, Function as RAREs in Yeast.
FIG. 13A, Sequences of the response elements used in this study consisting of a direct repeat of a hexamer sequence spaced by one to five base pairs (DR1-DR5), or an inverted repeat of the same hexamer with no spacing (IR0). The hexamer repeats are indicated by arrows and the spacer sequences are presented in lower case. FIG. 13B, Transactivation of URA3 reporter genes containing the different elements by homodimers and heterodimers of RARxcex1 and RXRxcex1. The sequences represented in FIG. 13A were cloned into the promoter of a URA3 reporter gene carried on a centromeric plasmid to generate the DRn-URA3 reporter series and IR0-URA3. The reporters were introduced into a yeast strain containing multicopy vectors expressing RARxcex1, RXRxcex1 or no receptor. Reporter activities in the presence or absence of ligand (500 nM) were determined by measuring the OMPdecase activity (Loison, G. et al., Yeast 5:497-507 (1989)). xe2x80x9cControlxe2x80x9d indicates the basal reporter activity in the absence of receptors (using xe2x80x9cemptyxe2x80x9d expression vectors), and the experiments using only one receptor were performed in the presence of the corresponding xe2x80x9cemptyxe2x80x9d vector. The white, hatched and black columns indicate the reporter activity in the presence of no ligand, all-trans RA and 9-cis RA, respectively. The reporter activities are given as units of OMPdecase activity per minute per mg protein, and the values represent the average of at least 2 experiments using at least 2 different clones per experiment. Deviation of values was less than 10%. FIG. 13C, Gel retardation assays using radiolabelled probes (containing the sequences described in 13A) and cell-free extracts from yeast coexpressing RARxcex1 and RXRxcex1 were performed as described previously (Heery, D. M. et al., Proc. Natl. Acad. Sci. USA 90:4281-4285 (1993)). Similar amounts of each probe (50,000 cpm) were used in the assays.
FIGS. 14A and B. Transactivation of the DR1-URA3 Reporter Gene by RXRxcex1 and RARxcex1/RXRxcex1 Heterodimers, Synergistic activation of the CRBPII RXRE-URA3 by RXRxcex1, and Effect of Deletions in RXRxcex1 on Reporter Activation.
FIG. 14A, Ligand dose responses of DR1 reporter activity in the presence of RXRxcex1 and RARxcex1/RXRxcex1 heterodimers. Activation of the DR1-URA3 reporter measured as OMPdecase activities in cell-free extracts of yeast expressing RXRxcex1, or RARxcex1 and RXRxcex1 together, grown in the presence of all-trans RA (empty circles) and 9-cis RA (filled circles) at the indicated concentrations, as described previously (Heery, D. M. et al., Proc. Natl. Acad. Sci. USA 90:4281-4285 (1993)). FIG. 14B, Activation of DR1-URA3 and CRBPII RXRE-URA3 reporters in yeast expressing RARxcex1, RXRxcex1, RXRxcex1xcex94B and dnRXRxcex1 or no receptor (control) in the presence or absence of ligand.
The present invention is based in part on the isolation of a DNA sequence encoding members of the RXR receptor family.
The present invention is additionally based on the novel observation that three types of nuclear receptors, RAR, RXR and TR, can form heterodimers at physiological conditions. The heterodimers thus formed are able to bind to a RE with a much greater efficiency than the respective homodimers.
Based on these observations, the present invention provides previously unknown DNA sequences as well as heterodimeric proteins.
The present invention discloses DNA sequences which encode 1 isoform of mRXR-xcex2 (Sequence ID No. 1), 1 isoform of hRXR-xcex2 (Sequence ID No. 3), 1 isoform of mRXR-xcex1 (Sequence ID No. 5), and 1 isoform of mRXR-xcex3 (Sequence ID No. 7). Using these sequences, or fragments thereof, as a probe, in conjunction with procedures known in the art, such as anchored PCR or blotting, it is now possible to isolate DNA sequences encoding homologous RXR receptors from other organisms, as well as isolating other subtypes of RXR, and other isotypes of the various subtypes of RXR.
As used herein, a xe2x80x9csubtypexe2x80x9d of RXR is identified by the presence of a subtype specific sequence which occurs within the A, B and/or D regions of the receptor. All isotypes from a given organism of a specific RXR family, for example all isoforms of human RXR-xcex2, possess a conserved sequence which defines the subtype within these regions.
As used herein, an xe2x80x9cisoformxe2x80x9d of a particular subtype of RXR receptor is identified by sequence heterogeneity which is present in the A region of the RXR receptor. Various isoforms of a RXR receptor from a given organism will possess differing A region sequences.
For example, one skilled in the art can use: the A, B, and/or D region of Sequence ID No. 10, which encodes mouse RXR-xcex3 specific regions of RXR, to isolate monkey RXR-xcex3 sequences, or the B and/or D regions of this sequence to isolate other isoforms of mouse (or any other organism) RXR-xcex3.
The present invention further includes cells or organisms transformed with the above sequences. One skilled in the art can readily transform prokaryotes, such as E. coli and B. subtilis, as well as eukaryotes, such as human cells, insect cells and yeast with the above sequences.
The present invention additionally discloses heterodimeric proteins comprised of two non-identical subunits. One of the subunits is either a RAR or TR, and the other subunit is a RXR.
The heterodimeric proteins of the present invention include, but are not limited to, proteins wherein the first subunit is a RAR selected from the group consisting of the isotypes of the RAR-xcex1, RAR-xcex2, RAR-xcex3, TR-xcex1 or TR-62  receptor families, and the second subunit is a RXR selected from the group consisting of the isotypes of the RXRxcex1, RXR-xcex2, or RXR-xcex3 receptor families.
The heterodimers of the present invention include, but are not limited to, proteins comprised of one subunit selected from the group consisting of: RAR-xcex11, RAR-xcex12, RAR-xcex13, RAR-xcex14, RAR-xcex15, RAR-xcex16, RAR-xcex17, RAR-xcex21, RAR-xcex22, RAR-xcex23, RAR-xcex24, RAR-xcex31, RAR-xcex32, RAR-xcex33, RAR-xcex34, RAR-xcex35, RAR-xcex36, RAR-xcex37, TR-xcex11, TR-xcex12, TR-xcex21, TR-xcex22; and the other subunit being selected from the group consisting of mRXR-xcex1 (Sequence ID No. 9), hRXR-xcex2 (Sequence ID No. 4), mRXR-xcex2 (Sequence ID No. 2), or mRXR-xcex3 (Sequence ID No. 8).
The present invention further provides highly purified subtypes and isoforms of RXR. Such purified RXR can exist as a monomer or a homodimer. As used herein, a protein is said to be highly purified if the protein possesses a specific activity that is greater than that found in whole cell extracts (WCE) containing the protein. For example, the specific activity commonly observed with WCE of HeLa cells is 156 cpm/ug (see Examples for assay conditions). The highly purified forms of RXR have a specific activity from about 1461 to 7,750,000 cpm/ug. Examples of the amino acid sequences of various highly purified RXR""s of the present invention are depicted in Sequence ID No. 2 (mRXR-xcex2), Sequence ID No. 4 (hRXR-xcex2), Sequence ID No. 6 (mRXR-xcex1), and Sequence ID No. 8 (mRXR-xcex3).
Any eukaryotic organism can be used as a source for the dimeric subunits, or the genes encoding same, as long as the source organism naturally contains such a subunit. As used herein, xe2x80x9csource organismxe2x80x9d refers to the original organism from which the amino acid or DNA sequence of the subunit is derived, regardless of the organism the subunit is expressed in or ultimately isolated from. For example, a human is said to be the xe2x80x9csource organismxe2x80x9d of RAR-xcex11 expressed in yeast as long as the amino acid sequence is that of human RAR-xcex11. The most preferred source organisms are human, mouse, and chicken.
A variety of methodologies known in the art can be utilized to obtain the subunits of the dimeric proteins of the present invention. In one embodiment, the subunits are purified from tissues or cells which naturally produce the given subunit. One skilled in the art can readily follow known methods for isolating proteins in order to obtain the desired subunit. These include, but are not limited to, immunochromotography, HPLC, size-exclusion chromatography, ion-exchange chromatography, and affinity chromatography.
The present invention further provides methods of purifying subtypes and isoforms of RXR. In detail, RXR""s can be purified by:
a) contacting a sample containing a RXR protein with a DEAE chromatography column in the presence of a buffer containing from about 50 mM KCl;
b) retrieving the RXR in the flow through fraction from the column;
c) contacting the flow through fraction (b) with a HEP-UG column;
d) eluting the RXR from the column using from about 290 mM KCl;
e) contacting the KCl eluted RXR (d) with a phenyl-5PW column;
f) eluting the RXR from the column using from about 250 mM ammonium sulfate;
g) contacting the ammonium sulfate eluted RXR (f) with a HEP-TSK column;
h) eluting the RXR from the column using from about 250 mM KCl;
i) contacting the KCl eluted RXR (h) with a HAP-TSK column; and
j) eluting the RXR from the column using from about 150 mM potassium phosphate.
The RXR""s obtained by the above method can be either monomeric or dimeric. One skilled in the art can readily adapt the above purification scheme to delete some or incorporate other purification steps.
In another embodiment, the subunits are purified from cells which have been altered to express the desired subunit.
As used herein, a cell is said to be xe2x80x9caltered to express a desired subunitxe2x80x9d when the cell, through genetic manipulation, is made to produce a protein which it normally does not produce, or which the cell normally produces at low levels. One skilled in the art can readily adapt procedures for introducing and expressing either genomic or cDNA sequences into either eukaryotic or prokaryotic cells, in order to generate a cell which produces a desired subunit.
There are a variety of source organisms for DNA encoding the desired subunit, including those subunits whose DNA sequence have been identified, such as the sequences found in Ruberte et al., Development 111:45-60 (1991), Chambon et al., Seminars in Dev. Biol. 2:153-159 (1991), Koelle et al., Cell 67:59-77 (1991), Mangelsdorf et al., Nature 345:224-229 (1990), Hamada et al., Proc. Natl. Acad. Sci. USA 86:8289-8293 (1989), Oro et al., Nature 347:298-301 (1990).
Alternatively, since probes are available which are capable of hybridizing to the various distinct subtypes and isoforms of RAR, RXR, and TR, DNA sequences encoding the desired subunits can be obtained by routine hybridization and selection from any host which possesses these receptors.
A nucleic acid molecule, such as DNA, is said to be xe2x80x9ccapable of expressingxe2x80x9d a polypeptide if it contains nucleotide sequences which contain transcriptional and translational regulatory information and such sequences are xe2x80x9coperably linkedxe2x80x9d to nucleotide sequences which encode the polypeptide. An operable linkage is a linkage in which the regulatory DNA sequences and the DNA sequence sought to be expressed are connected in such a way as to permit gene sequence expression. The precise nature of the regulatory regions needed for gene sequence expression may vary from organism to organism, but shall in general include a promoter region which, in prokaryotes, contains both the promoter (which directs the initiation of RNA transcription) as well as the DNA sequences which, when transcribed into RNA, will signal the initiation of RXR synthesis. Such regions will normally include those 5xe2x80x2-non-coding sequences involved with initiation of transcription and translation, such as the TATA box, capping sequence, CAAT sequence, and the like.
If desired, the non-coding region 3xe2x80x2 to the gene sequence encoding RXR may be obtained by the above-described methods. This region may be retained for its transcriptional termination regulatory sequences, such as termination and polyadenylation. Thus, by retaining the 3xe2x80x2-region naturally contiguous to the DNA sequence encoding RXR, the transcriptional termination signals may be provided. Where the transcriptional termination signals are not satisfactorily functional in the expression host cell, then a 3xe2x80x2 region functional in the host cell may be substituted.
Two DNA sequences (such as a promoter region sequence and the RXR encoding sequence) are said to be operably linked if the nature of the linkage between the two DNA sequences does not (1) result in the introduction of a frame-shift mutation, (2) interfere with the ability of the promoter region sequence to direct the transcription of the RXR gene sequence, or (3) interfere with the ability of the RXR gene sequence to be transcribed by the promoter region sequence. Thus, a promoter region would be operably linked to a DNA sequence if the promoter were capable of effecting transcription of that DNA sequence.
Thus, to express the RXR, transcriptional and translational signals recognized by an appropriate host are necessary.
The present invention encompasses the expression of the RXR proteins (or a functional derivative thereof) and the dimeric proteins of the present invention in either prokaryotic or eukaryotic cells. Preferred prokaryotic hosts include bacteria such as E. coli, Bacillus, Streptomyces, Pseudomonas, Salmonella, Serratia, etc. The most preferred prokaryotic host is E. coli. Bacterial hosts of particular interest include E. coli K12 strain 294 (ATCC 31446), E. coli X1776 (ATCC 31537), E. coli W3110 (Fxe2x88x92, lambdaxe2x88x92, prototrophic (ATCC 27325)), and other enterobacterium such as Salmonella typhimurium or Serratia marcescens, and various Pseudomonas species. Under such conditions, the RXR will not be glycosylated. The procaryotic host must be compatible with the replicon and control sequences in the expression plasmid.
To express RXR (or a functional derivative thereof) in a prokaryotic cell (such as, for example, E. coli, B. subtilis, Pseudomonas, Streptomyces, etc.), it is necessary to operably link the RXR coding sequence to a functional prokaryotic promoter. Such promoters may be either constitutive or, more preferably, regulatable (i e., inducible or derepressible). Examples of constitutive promoters include the int promoter of bacteriophage xcex, the bla promoter of the xcex2-lactamase gene sequence of pBR322, and the CAT promoter of the chloramphenicol acetyl transferase gene sequence of pPR325, etc. Examples of inducible prokaryotic promoters include the major right and left promoters of bacteriophage xcex (PL and PR), the trp, recA, lacZ, lac1, and gal promoters of E. coli, the xcex1-amylase (Ulmanen, I. et al., J. Bacteriol. 162:176-182 (1985)) and the xc3xa7-28-specific promoters of B. subtilis (Gilman, M. Z. et al., Gene sequence 32:11-20 (1984)), the promoters of the bacteriophages of Bacillus (Gryczan, T. J., In: The Molecular Biology of the Bacilli, Academic Press, Inc., NY (1982)), and Streptomyces promoters (Ward, J. M. et al., Mol. Gen. Genet. 203:468-478 (1986)).
Prokaryotic promoters are reviewed by Glick, B. R., (J. Ind. Microbiol. 1:277-282 (1987)); Cenatiempo, Y. (Biochimie 68:505-516 (1986)); and Gottesman, S. (Ann. Rev. Genet. 18:415-442 (1984)).
Proper expression in a prokaryotic cell also requires the presence of a ribosome binding site upstream of the gene sequence-encoding sequence. Such ribosome binding sites are disclosed, for example, by Gold, L. et al. (Ann. Rev. Microbiol. 35:365-404 (1981)).
Preferred eukaryotic hosts include yeast, fungi, insect cells, mammalian cells either in vivo, or in tissue culture. Mammalian cells which may be useful as hosts include HeLa cells, cells of fibroblast origin such as VERO or CHO-K1, or cells of lymphoid origin, such as the hybridoma SP2/O-AG14 or the myeloma P3x63Sg8, and their derivatives. Preferred mammalian host cells include SP2/0 and J558L, as well as neuroblastoma cell lines such as IMR 332 that may provide better capacities for correct post-translational processing.
For a mammalian host, several possible vector systems are available for the expression of the RXR proteins or the dimers herein described. A wide variety of transcriptional and translational regulatory sequences may be employed, depending upon the nature of the host. The transcriptional and translational regulatory signals may be derived from viral sources, such as adenovirus, bovine papilloma virus, Simian virus, or the like, where the regulatory signals are associated with a particular gene sequence which has a high level of expression. Alternatively, promoters from mammalian expression products, such as actin, collagen, myosin, etc., may be employed. Transcriptional initiation regulatory signals may be selected which allow for repression or activation, so that expression of the gene sequences can be modulated. Of interest are regulatory signals which are temperature-sensitive so that by varying the temperature, expression can be repressed or initiated, or are subject to chemical (such as metabolite) regulation.
Yeast provides substantial advantages in that it can also carry out post-translational peptide modifications. A number of recombinant DNA strategies exist which utilize strong promoter sequences and high copy number of plasmids which can be utilized for production of the desired proteins in yeast. Yeast recognizes leader sequences on cloned mammalian gene sequence products and secretes peptides bearing leader sequences (i.e., pre-peptides).
Any of a series of yeast gene sequence expression systems incorporating promoter and termination elements from the actively expressed gene sequences coding for glycolytic enzymes produced in large quantities when yeast are grown in media rich in glucose can be utilized. Known glycolytic gene sequences can also provide very efficient transcriptional control signals. For example, the promoter and terminator signals of the phosphoglycerate kinase gene sequence can be utilized. Yeast is an especially preferred host since yeast cells do not contain RA receptors.
Another preferred host is insect cells, for example the Drosophila larvae. Using insect cells as hosts, the Drosophila alcohol dehydrogenase promoter can be used (Rubin, G. M., Science 240:1453-1459 (1988)). Alternatively, baculovirus vectors can be engineered to express large amounts of the RXR in insects cells (Jasny, B. R., Science 238:1653 (1987); Miller, D. W. et al., in Genetic Engineering (1986), Setlow, J. K. et al., eds., Plenum, Vol. 8, pp. 277-297).
As discussed above, expression of RXR in eukaryotic hosts requires the use of eukaryotic regulatory regions. Such regions will, in general, include a promoter region sufficient to direct the initiation of RNA synthesis. Preferred eukaryotic promoters include the promoter of the mouse metallothionein I gene sequence (Hamer, D. et al., J. Mol. Appl. Gen. 1:273-288 (1982)); the TK promoter of Herpes virus (McKnight, S., Cell 31:355-365 (1982)); the SV40 early promoter (Benoist, C. et al., Nature (London) 290:304-310 (1981)); the yeast gal4 gene sequence promoter (Johnston, S. A. et al., Proc. Natl. Acad. Sci. (USA) 79:6971-6975 (1982); Silver, P. A. et al., Proc. Natl. Acad. Sci. (USA) 81:5951-5955 (1984)).
As is widely known, translation of eukaryotic mRNA is initiated at the codon which encodes the first methionine. For this reason, it is preferable to ensure that the linkage between a eukaryotic promoter and a DNA sequence which encodes the RXR (or a functional derivative thereof) does not contain any intervening codons which are capable of encoding a methionine (i.e., AUG). The presence of such codons results either in a formation of a fusion protein (if the AUG codon is in the same reading frame as the RXR coding sequence) or a frame-shift mutation (if the AUG codon is not in the same reading frame as the RXR coding sequence).
The RXR coding sequence and an operably linked promoter may be introduced into a recipient prokaryotic or eukaryotic cell either as a non-replicating DNA (or RNA) molecule, which may either be a linear molecule or, more preferably, a closed covalent circular molecule. Since such molecules are incapable of autonomous replication, the expression of the RXR may occur through the transient expression of the introduced sequence. Alternatively, permanent expression may occur through the integration of the introduced sequence into the host chromosome.
In one embodiment, a vector is employed which is capable of integrating the desired gene sequences into the host cell chromosome. Cells which have stably integrated the introduced DNA into their chromosomes can be selected by also introducing one or more markers which allow for selection of host cells which contain the expression vector. The marker may provide for prototrophy to an auxotrophic host, biocide resistance, e.g., antibiotics, or heavy metals, such as copper, or the like. The selectable marker gene sequence can either be directly linked to the DNA gene sequences to be expressed, or introduced into the same cell by co-transfection. Additional elements may also be needed for optimal synthesis of single chain protein-binding mRNA. These elements may include splice signals, as well as transcription promoters, enhancers, and termination signals. cDNA expression vectors incorporating such elements include those described by Okayama, H., Molec. Cell. Biol. 3:280 (1983).
In a preferred embodiment, the introduced sequence will be incorporated into a plasmid or viral vector capable of autonomous replication in the recipient host. Any of a wide variety of vectors may be employed for this purpose. Factors of importance in selecting a particular plasmid or viral vector include: the ease with which recipient cells that contain the vector may be recognized and selected from those recipient cells which do not contain the vector; the number of copies of the vector which are desired in a particular host; and whether it is desirable to be able to xe2x80x9cshuttlexe2x80x9d the vector between host cells of different species. Preferred prokaryotic vectors include plasmids such as those capable of replication in E. coli (such as, for example, pBR322, ColE1, pSC101, pACYC 184, xcfx80VX. Such plasmids are, for example, disclosed by Maniatis, T. et al. (In: Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1982)). Bacillus plasmids include pC194, pC221, pT127, etc. Such plasmids are disclosed by Gryczan, T. (In: The Molecular Biology of the Bacilli, Academic Press, NY (1982), pp. 307-329). Suitable Streptomyces plasmids include pIJ101 (Kendall, K. J. et al., J. Bacteriol. 169:4177-4183 (1987)), and streptomyces bacteriophages such as xcfx86C31 (Chater, K. F. et al., In: Sixth International Symposium on Actinomycetales Biology, Akademiai Kaido, Budapest, Hungary (1986), pp. 45-54). Pseudomonas plasmids are reviewed by John, J. F. et al. (Rev. Infect. Dis. 8:693-704 (1986)), and Izaki, K. (Jpn. J. Bacteriol. 33:729-742 (1978)).
Preferred eukaryotic plasmids include BPV, vaccinia, SV40, 2-micron circle, etc., or their derivatives. Such plasmids are well known in the art (Botstein, D. et al., Miami Wntr. Symp. 19:265-274 (1982); Broach, J. R., In: The Molecular Biology of the Yeast Saccharomyces: Life Cycle and Inheritance, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., p. 445-470 (1981); Broach, J. R., Cell 28:203-204 (1982); Bollon, D. P. et al., J. Clin. Hematol. Oncol. 10:39-48 (1980); Maniatis, T., In: Cell Biology: A Comprehensive Treatise, Vol. 3, Gene sequence Expression, Academic Press, NY, pp. 563-608 (1980)).
Once the vector or DNA sequence containing the construct(s) has been prepared for expression, the DNA construct(s) may be introduced into an appropriate host cell by any of a variety of suitable means: transformation, transfection, conjugation, protoplast fusion, electroporation, calcium phosphate-precipitation, direct microinjection, etc. After the introduction of the vector, recipient cells are grown in a selective medium, which selects for the growth of vector-containing cells. Expression of the cloned gene sequence(s) results in the production of RXR, or fragments thereof. This can take place in the transformed cells as such, or following the induction of these cells to differentiate (for example, by administration of bromodeoxyuracil to neuroblastoma cells or the like).
A cell can be altered to express either a single subunit of the dimer, or altered to express both subunits of the dimer. When the cell is altered to express a single subunit, the heterodimers of the present invention are generated by mixing the individual subunits which have isolated from two different transformed hosts while the homodimers are generated by incubating the monomeric subunit under conditions which promote dimerization.
A variety of incubation conditions can be used to form the dimers of the present invention. The most preferred conditions are those which mimic physiological conditions. In the examples provided below, heterodimers were formed in 150 mM KCL.
When the cells are altered to express both subunits, the dimer can be purified from the single host.
The dimeric proteins of the present invention can be used in a variety of procedures and methods, such as for the generation of antibodies, for use in identifying pharmaceutical compositions, and for studying DNA/protein interaction.
In one embodiment, the dimer is used as an immunogen to generate an antibody which is capable of binding to the dimer. In a further aspect of this embodiment, the antibody is additionally incapable of binding to the individual subunit even though it binds to the dimer.
Any of the dimers of the present invention can be used to produce antibodies or hybridomas. One skilled in the art will recognize that if an antibody is desired that will bind to hRAR-xcex1/hRXR-xcex1, such a dimer would be generated as described above and used as an immunogen. The resulting antibodies are then screened for the ability to bind the dimer. Additionally, the antibody can be screened for it""s inability to bind the individual subunits.
The antibodies of the present invention include monoclonal and polyclonal antibodies, as well fragments of these antibodies, and humanized forms. Humanized forms of the antibodies of the present invention may be generated using one of the procedures known in the art such as chimerization or CDR grafting.
The invention also provides hybridomas which are capable of producing the above-described antibodies. A hybridoma is an immortalized cell line which is capable of secreting a specific monoclonal antibody.
In general, techniques for preparing monoclonal antibodies and hybridomas are well known in the art (Campbell, A. M., xe2x80x9cMonoclonal Antibody Technology: Laboratory Techniques in Biochemistry and Molecular Biology,xe2x80x9d Elsevier Science Publishers, Amsterdam, The Netherlands (1984); St. Groth et al., J. Immunol. Methods 35:1-21 (1980)).
Any animal (mouse, rabbit, etc.) which is known to produce antibodies can be immunized with the selected polypeptide. Methods for immunization are well known in the art. Such methods include subcutaneous or intraperitoneal injection of the polypeptide. One skilled in the art will recognize that the amount of polypeptide used for immunization will vary based on the animal which is immunized, the antigenicity of the polypeptide and the site of injection.
The polypeptide may be modified or administered in an adjuvant in order to increase the peptide antigenicity. Methods of increasing the antigenicity of a polypeptide are well known in the art. Such procedures include coupling the antigen with a heterologous protein (such as globulin or xcex2-galactosidase) or through the inclusion of an adjuvant during immunization.
For monoclonal antibodies, spleen cells from the immunized animals are removed, fused with myeloma cells, such as SP2/0-Ag14 myeloma cells, and allowed to become monoclonal antibody producing hybridoma cells.
Any one of a number of methods well known in the art can be used to identify the hybridoma cell which produces an antibody with the desired characteristics. These include screening the hybridomas with an ELISA assay, western blot analysis, or radioimmunoassay (Lutz et al., Exp. Cell Res. 175:109-124 (1988)).
Hybridomas secreting the desired antibodies are cloned and the class and subclass is determined using procedures known in the art (Campbell, A. M., Monoclonal Antibody Technology: Laboratory Techniques in Biochemistry and Molecular Biology, Elsevier Science Publishers, Amsterdam, The Netherlands (1984)).
For polyclonal antibodies, antibody containing antisera is isolated from the immunized animal and is screened for the presence of antibodies with the desired specificity using one of the above-described procedures.
In another embodiment of the present invention, the above-described antibodies are detectably labelled. Antibodies can be detectably labelled through the use of radioisotopes, affinity labels (such as biotin, avidin, etc.), enzymatic labels (such as horse radish peroxidase, alkaline phosphatase, etc.) fluorescent labels (such as FITC or rhodamine, etc.), paramagnetic atoms, etc. Procedures for accomplishing such labelling are well-known in the art, for example see (Sternberger, L. A. et al., J. Histochem. Cytochem. 18:315 (1970); Bayer, E. A. et al., Meth. Enzym. 62:308 (1979); Engval, E. et al., Immunol. 109:129 (1972); Goding, J. W. J. Immunol. Meth. 13:215 (1976)). The labeled antibodies of the present invention can be used for in vitro, in vivo, and in situ assays to identify cells or tissues which express a specific heterodimer.
In another embodiment of the present invention the above-described antibodies are immobilized on a solid support. Examples of such solid supports include plastics such as polycarbonate, complex carbohydrates such as agarose and sepharose, acrylic resins and such as polyacrylamide and latex beads. Techniques for coupling antibodies to such solid supports are well known in the art (Weir, D. M. et al., xe2x80x9cHandbook of Experimental Immunologyxe2x80x9d 4th Ed., Blackwell Scientific Publications, Oxford, England, Chapter 10 (1986); Jacoby, W. D. et al., Meth. Enzym. 34 Academic Press, N.Y. (1974)). The immobilized antibodies of the present invention can be used for in vitro, in vivo, and in situ assays as well as in immunochromotography.
In another embodiment of the present invention, methods of determining the expression of a specific dimer in a test sample are presented.
In detail, the methods comprise incubating a test sample with one or more of the antibodies of the present invention and assaying whether the antibody binds to the test sample.
Conditions for incubating an antibody with a test sample vary. Incubation conditions depend on the format employed in the assay, the detection methods employed, and the type and nature of the antibody used in the assay. One skilled in the art will recognize that any one of the commonly available immunological assay formats (such as radioimmunoassays, enzyme-linked immunosorbent assays, diffusion based Ouchterlony, or rocket immunofluorescent assays) can readily be adapted to employ the antibodies of the present invention. Examples of such assays can be found in Chard, T. xe2x80x9cAn Introduction to Radioimmunoassay and Related Techniquesxe2x80x9d Elsevier Science Publishers, Amsterdam, The Netherlands (1986); Bullock, G. R. et al., xe2x80x9cTechniques in Immunocytochemistry,xe2x80x9d Academic Press, Orlando, Fla. Vol. 1 (1982), Vol. 2 (1983), Vol. 3 (1985); Tijssen, P., xe2x80x9cPractice and Theory of Enzyme Immunoassays: Laboratory Techniques in Biochemistry and Molecular Biology,xe2x80x9d Elsevier Science Publishers, Amsterdam, The Netherlands (1985).
The test samples of the present invention include cells, protein or membrane extracts of cells, or biological fluids such as blood, serum, plasma, or urine. The test sample used in the above-described method will vary based on the assay format, nature of the detection method and the tissues, cells or extracts used as the sample to be assayed. Methods for preparing protein extracts or membrane extracts of cells are well known in the art and can be readily be adapted in order to obtain a sample which is capable with the system utilized.
In another embodiment of the present invention, methods are provided for identifying agents which are capable of binding to one of the dimeric proteins of the present invention.
In detail, said method comprises:
(a) contacting an agent with one or more of the dimeric proteins of the present invention; and
(b) determining whether the agent binds to the dimer.
In performing such an assay, one skilled in the art will be able to determine which isotype or subtype of a specific nuclear receptor an agent binds to, and hence determine what specific receptor(s) are utilized by the compound.
The agents screened in the above assay can be, but are not limited to, peptides, carbohydrates, and vitamin derivatives. The agent can be selected, and screened at random, rationally selected or rationally designed using protein modeling techniques.
For random screening, agents such as peptides, carbohydrates, or derivatives of RA, are selected at random and are assayed for there ability to bind to one of the heterodimers of the present invention using either direct or indirect methods.
Alternatively, agents may be rationally selected. As used herein, an agent is said to be xe2x80x9crationally selectedxe2x80x9d when the agent is chosen based on the physical structure of a known ligand of the heterodimer. For example, assaying compounds possessing a retinol like structure would be considered a rational selection since retinol like compounds will bind to a variety of the heterodimers.
Since highly purified dimers are now available, X-ray crystallography and NMR-imaging techniques can be used to identify the structure of the ligand binding site present on the heterodimer. Utilizing such information, computer modeling systems are now available that allows one to xe2x80x9crationally designxe2x80x9d an agent capable of binding to a defined structure (Hodgson, Biotechnology 8:1245-1247 (1990)), Hodgson, Biotechnology 9:609-613 (1991)).
As used herein, an agent is said to be xe2x80x9crationally designedxe2x80x9d if it is selected based on a computer model of the ligand binding site of the heterodimer.
In one aspect of the above-described binding assay, the assay is performed in the presence of a segment of DNA which has been identified as a RE. In this fashion agents can be identified which are capable of either stimulating or inhibiting the binding of the dimer to the RE. Any length of DNA can be used in such an assay as long as it contains at least one RE sequence.
In another embodiment, the above assay is performed in the absence of a RE. In this fashion, agents can be identified which bind to the dimer independently of DNA binding.
Further, the above assay can be modified so that it is capable of identifying agents which activated transcription of DNA sequences controlled by a RE.
In detail a cell or organism, such as a yeast cell, is altered using routine methods such that it expresses one or more of the RAR/RXR hetero- or homodimers of the present invention.
In one application, the cell is further altered to contain a RE, such as DR1, operably linked to a reporter sequence, such as luciferase, beta galactosidase, chloramphenicol acyltransferase or a selectable marker such as URA3. An agent is then incubated with the cell or organism and the expression of the reporter sequence or selectable marker activity is then assayed. By utilizing the above procedure, agents capable of stimulating RAR/RXR/RE dependent transcription or inhibiting ligand-induced transcription can be identified.
The present invention further discloses that RAR/RXR heterodimers, and RAR or RXR homodimers when expressed in yeast cells, are capable of transactivating the transcription of a sequence operably linked to an RARE. As described in Example 2, yeast cells do not naturally contain RAR and RXR receptors. Further, unlike most other eukaryotic organisms, yeasts, in general, do not appear to contain enzymes which interconvert different classes of retinoids. For example mammals contain enzymes (RA isomerase activities) which convert all-trans retinoic acid to 9-cis retinoic acid whereas yeast does not. In particular, yeast cells containing a chimeric RXR (RXR-ER.CAS) activate transcription in the presence of 9cis-RA, but not all-trans RA (Heery et al., PNAS 90:4281-4285 (1993)), and may be used to clone the mammalian all-trans-9cis isomerase by complementation with cDNA expression libraries made from mammalian cell-derived RNA. Other enzymes involved in RA metabolism might be similarly cloned by complementation, using positive and negative selection techniques. Therefore, yeast cells such as Saccharomyces cerevisiae are an especially preferred organism for expressing RAR and RXR receptor proteins and for using such an expression system to further study and identify agents which modulate RAR/RXR/RARE dependent transcription.
In general, a modified yeast cell is generated for use in studying transactivation by modifying the yeast cell, using routine genetic manipulations, such that it contains, and is capable of expressing, one or more subtypes or isoforms of RAR and/or RXR receptors , for example a cell is modified such that it expresses RARxcex11 and RXRxcex1 (see Example 2) or RXR (Example 3). The cell is then further modified so that a gene encoding a selectable marker activity, for example URA3 if the original yeast host is ura3, or a gene encoding an assayable marker activity, for example beta-galactosidase, is placed under the control of a RARE, for example a DR5 sequence.
Such a modified yeast cell is then used to identify agents which are capable of transactivating the particular RAR/RXR/RARE combination. In detail, when the above described modified yeast cell is incubated with an agent which is capable of binding to the RAR/RXR dimer, stimulating the dimer""s ability to bind to and activate the transcription of sequences linked to the RARE (transactivation), the cell will be capable of growth in a media not containing uracil, or can be identified as expressing the marker activity. By generating a variety of modified yeast cells, each one expressing a different RAR/RXR heterodimer or RAR or RXR homodimer and each containing one or more selectable markers linked to various RAREs, dimer isoform and subtype specific as well as RARE specific activating agents can be identified.
In other applications, the yeast system described above and herein, can be further utilized to 1) identify RARE sequences, 2) identify antagonists and agonist of transcription which is controlled by RAR/RXR heterodimers or RAR or RXR homodimers, 3) identify and clone genes encoding enzymes capable of metabolising retinoids (i.e., enzymes with RA isomerase activity), and 4) to identify and clone genes encoding novel dimeric partners of RXRs by complementation with mammalian RNA-derived yeast expression vectors.
Specifically, to identify DNA sequences which act as an RARE, for example sequences such as DR1, DR2 and DR5, a yeast cell is modified as such that the cell expresses one or more RAR/RXR hetero- or homodimers. The cell is further modified such that the cell contains the DNA sequence which is to be tested. The sequence which is to be tested for RARE activity is placed 5xe2x80x2 to a gene encoding selectable marker activity, for example, URA3 in a ura3 host cell, or a gene encoding an assayable marker activity. The cell is then incubated in the presence of an agent which is known to activate RAR/RXR, RAR or RXR dependent transactivation. Cells in which the DNA sequence placed upstream to the selectable marker contain an RARE activity will be capable of growing in a selection media devoid of uracil or will express the assayable activity encoded by the marker gene. Such a procedure can be utilized for screening randomly cloned DNA sequences, a shot-gun type approach, or can be used to test DNA sequences which are rationally designed based on the sequence of known RAREs.
To identify antagonists of a specific RAR/RXR hetero- or homodimer, a yeast cell is modified such that the cell expresses one or more RAR/RXR hetero- or homodimers. The cell is further modified such that the cell contains a RARE placed 5xe2x80x2 to a lethal marker, the expression of such a marker leads to cell death. The modified yeast cell is then incubated in the presence of 2 agents, the first agent being a compound which is known to transactivate the specific RAR/RXR/RARE combination contained in the yeast cell, the second agent being the compound which is being tested for antagonistic activity. Agents which act as a antagonist will prevent the expression of the lethal gene.
To identify DNA sequences which encode enzymes capable of metabolising various retinoids, for example the enzyme responsible for isomerisation of all-trans retinoic acid to 9 cis-retinoic acid in a mammal, a yeast cell is modified such that it expresses one or more RAR/RXR hetero- or homodimers. The cell is further modified such that it contains a RARE placed 5xe2x80x2 to a gene encoding a selectable marker activity, for example, URA3 in a ura3 host cell, or gene encoding an assayable marker activity. The cell is then used as a host for expressing cDNA""s which have been isolated from a mammal (or other source) which is known or suspected of being capable of interconverting a specific class of retinoids, for example a human skin cDNA library.
After transformation with the cDNA library, the yeast cell is then incubated with an agent which does not transactivate the specific RAR/RXR/RARE combination contained within the cell unless the agent is first converted into an active form. Cells capable of growth in a selection media, or cells which express the assayable marker, are supposed to contain gene sequences encoding an enzyme which converts the retinoid into an active form. Alternatively, the cell can be incubated with a retinoid which is capable of transactivating the RAR/RXR/RARE combination contained in the cell but is incapable of transactivation once the agent has been converted into an inactive form.
The above procedure can be modified to use nuclear and/or cytosolic extracts from the altered cell containing the hetero- or homodimer, as opposed to using the intact cells. In such an application an extract of a cell expressing a RAR/RXR heterodimer, or a RAR or RXR homodimer, is mixed with an expression module containing an RARE operably linked to a reporter sequence. The extract/expression module is then incubated with an agent and the expression of the reporter sequence is assayed.
In another embodiment of the present invention, kits are provided which contain the necessary reagents to carry out the previously described assays.
Specifically, the invention provides a compartmentalized kit to receive, in close confinement, one or more containers which comprises: (a) a first container comprising one of the antibodies of the present invention, one or more of the dimers of the present invention, or one or more of the modified cells of the present invention; and (b) one or more other containers comprising one or more of the following: wash reagents, reagents capable of detecting presence of bound antibodies or heterodimers from the first container, RE sequences, or antagonist on agonists RAR/RXR/RARE transactivation.
In detail, a compartmentalized kit includes any kit in which reagents are contained in separate containers. Such containers include small glass containers, plastic containers or strips of plastic or paper. Such containers allow one to efficiently transfer reagents from one compartment to another compartment such that the samples and reagents are not cross-contaminated, and the agents or solutions of each container can be added in a quantitative fashion from one compartment to another. Such containers will include a container which will accept the test sample, a container which contains the antibodies used in the assay, containers which contain wash reagents (such as phosphate buffered saline, Tris-buffers, etc.), and containers which contain the reagents used to detect the bound antibody.
Types of detection reagents include labelled secondary antibodies, or in the alternative, if the primary antibody is labelled, the chromophoric, enzymatic, or antibody binding reagents which are capable of reacting with the labelled antibody. One skilled in the art will readily recognize that the disclosed antibodies of the present invention can readily be incorporated into one of the established kit formats which are well known in the art.
The present invention further provides methods of identifying and isolating DNA sequences which bind to the dimers of the present invention. Specifically, the dimers of the present invention can be used to isolate or screen given sequences for the ability to be bound by the dimer or act as an RARE.
There are a variety of methods known in the art for isolating sequences which are bound by a protein. In one such method, the dimer is immobilized on a solid support and used as an affinity matrix to isolate sequences which bind to it (Arcangioli et al., Eur. J. Biochem. 179:3459-364 (1989)).
For example, a hRAR-xcex11/hRXR-xcex2 heterodimer, or a RXRxcex1 homodimer, is immobilized on sepharose and sheared human DNA (most preferably 20bp-2kb in length) is washed over the column. Sequences which bind to the dimer will stick to the column whereas sequences which don""t will be removed in the washes. Additionally, the bound sequences can be amplified using PCR prior to cloning.
Alternatively, the dimeric protein can be used to screen a genomic library whose DNA has been immobilized on a solid support such as nitrocellulose. (Sharp et al., Biochem. Biophys Acta 1048:306-309 (1990); Walker et al., Nuc. Acid. Res. 18:1159-1166 (1990)).
The present invention further provides methods of regulating gene expression in a cell.
In detail, a cell can be altered such that it contains a DNA sequence operably linked to an RE. Additionally, the cell can be altered to express various subunits which form the dimeric proteins of the present invention. By selecting the appropriate subunit/RE combination, one skilled in the art can generate a cell which expresses a given sequence in response to a particular agent.